The goal of this research is to inactivate single, preselected genes in cultured human cells and determine if the loss of their function results in sensitivity to ionizing radiation. Studies on the molecular-genetic basis of radiosensitivity in humans have thus far been limited to examination of cultured human cells from patients with heritable diseases that confer a radiosensitive phenotype, such as Ataxia telangiectasia. This approach may eventually lead to the molecular cloning and characterization of a few genes. On the other hand, experimental systems based on mutant rodent cell lines and lower eucaryotes are yielding genes that influence radiosensitivity and have human homologues, however, the role of these genes in human cells is not known. This proposal seeks to develop procedures to test these genes for functionality in human cells. Individual, preselected genes will be inactivated in cultured human cells by targeting their transcripts for destruction by ribozymes. The key aims in this proposal are to design effective ribozymes and to develop a practical means of expressing them in an intracellular environment. Once this technology is in place it will be straightforward to create loss of function mutants for any gene in question in cultured human cells. These cells will then be exposed to ionizing radiation and analyzed for their ability to survive, and correctly repair radiation-damaged DNA. Hopefully the results of this research will enhance our understanding of the molecular mechanisms that influence the outcome of exposure of human cells to ionizing radiation. Also, this approach may identify genes that have clinical relevance as tumor suppressor genes or as predictors of patient radiosensitivity.